Expanding the throughput of real-time toxicological screening of cardiac differentiation by expressing a synthetic luciferase/luciferin genetic pathway in iPSCs Project Summary. This Small Business Innovation Research (SBIR) Phase I project proposes to develop autonomously bioluminescent induced pluripotent stem cells (iPSCs) for continuous, reagent- free, and real-time toxicological screening to address the National Institute of Environmental Health Sciences (NIEHS) request for novel high-throughput assays to evaluate the effects of chemical compounds on the differentiation of pluripotent stem cells, as their ability to differentiate along well- defined lineage pathways offers a powerful approach to understanding how chemical perturbations disrupt metabolic and regulatory functions along those pathways. The autobioluminescent iPSCs developed here will significantly contribute towards NIEHS's mission to 'discover how the environment affects people in order to promote healthier lives' by expanding the knowledge base of chemical exposure toxicological effects. This is especially important given that the commercial marketplace maintains an inventory of tens of thousands of chemicals, the majority of which are poorly understood in terms of their risks and hazards to human health, and currently require animal-based testing approaches that are expensive, time consuming, and ethically contentious to determine their human health effects. As an alternative, stem cell-based assays such as the one developed here may mimic human disease states more reliably than animal models while providing valuable information towards understanding how chemical exposures influence cancer risks, developmental defects, and other adverse health outcomes. Therefore, there exists significant impetus for the integration of stem cells in chemical screening programs such as Tox21 and ToxCast, but under the mandate that they function under high-throughput conditions. While this goal is not obtainable using existing bioluminescent reporter technologies such as firefly luciferase that must be provided with a chemical substrate to activate their light emission responses, resulting in only marginally informative single time point snapshots of potential toxicological interactions, 490 BioTech's synthetic luciferase technology enables reporter cells to emit light continuously and in real- time, thereby providing an uninterrupted stream of visual data over the lifetime of the cell as it interacts and reacts to chemical perturbations. The goal of this research effort is o express our synthetic luciferase system in iPSCs and demonstrate real-time, continuous visualization of iPSC to cardiomyocyte differentiation under chemical toxicity exposure pressures. Our specific aims will focus on assay development and optimization, benchmark comparisons against existing commercial assay systems, and assay validation against a chemical subset of the Tox21 10K library.